It is now well established that the parameters of GR-mediated gene induction (Amax, EC50, and PAA) can be modulated by changing the concentrations of involved factors. Most of these studies have been conducted by looking at the activity of a single saturating or subsaturating concentration of agonist, occasionally with a saturating concentration of antisteroid, in transiently transfected tissue culture cells. Our recent studies in human peripheral mononuclear cells (PBMCs) have confirmed that changes in factor concentration affect the induction parameters of endogenous, as well as exogenous, GR-regulated genes. These results provide strong support for our hypothesis that the modulation of GR induction parameters is a relevant feature of human physiology. The number of reported cofactors involved in steroid-regulated gene expression that can modifiy Amax, EC50, and PAA is large and growing. Current assays determine the temporal ordering of cofactor binding to DNA. However, no method currently exists to elucidate the temporal ordering of the biological function of cofactors. This is because cofactor binding to DNA is not equivalent to cofactor action. Thus, two critical, unanswered questions in studies of the role of cofactors in receptor-mediated gene transcription are (i) the step at which each cofactor exerts its biological activity and (ii) the precise nature of that activity. Many factors are described operationally in terms of whether the Amax is increased or decreased with changing concentrations of cofactor. This causes considerable confusion, though, when the same cofactor increases Amax in one system and decreases it in another. Furthermore, a partial inhibitor can masquerade as an activator to increase the Amax by giving a higher response if it diverts the reaction sequence output to a higher yielding pathway. In an effort to eliminate the confusion resulting from observational definitions, and to better understand how cofactors act, we have continued our collaboration with Carson Chow to extend our recently developed, experimentally supported mathematical theory of steroid hormone action. We find that this theory can deduce both the kinetic properties of factor action, which are independent of whether Amax increases or decreases, and the position in the reaction sequence at which a cofactor acts relative to a concentration limiting step (CLS). Further analysis of the underlying equations revealed that the analysis also applies to reactions involving the competition of any two factors, in which case even more information can be obtained. With two cofactors, the nature and location of action of both cofactors can be determined simultaneously relative not only to the CLS but also to each other using graphical methods similar to those used in enzyme kinetics. Even if a precise mechanistic interpretation is not possible, differences in the graphical analyses can reveal mechanistic non-equivalence of two cofactors that otherwise may be thought to share a common mode of action. This methodology has been validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new graphical analysis tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available. It is, therefore, now possible to construct an ordered sequence of events based on the biological function of cofactors, much as in the ordering of pathways by epistasis analysis, even if the biochemical properties of the cofactors are not known. The above studies have provided previously unobtainable molecular information both about the determinants of glucocorticoid steroid activity and about the modulation of the total activity (Amax) and dose-response curve (EC50) of agonists. They also constitute a rational approach to identifying inhibitors of cofactors at specific steps in steroid hormone action. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.